The objective of the proposed studies is to better understand the fundamental mechanisms that determine the NMR relaxation properties of protons in tissues and the changes that occur in disease. The project will extend the methods developed in the previously funded period and pursue measurements to test specific hypotheses. Contrast in NMR images arises primarily from the heterogenous distribution of tissue relaxation properties but no adequate model exists to quantitatively account for the relaxation rates, especially 1/T2, even of normal tissues. There are, for example, major differences between the properties of macromolecules in solution and those of organized tissue. These differences include the magnitudes of cross-relaxation rates, chemical exchange and other T2 shortening phenomena such as diffusion effects and the importance of anisotropic motions of oriented water molecules. We will quantify the contributions of individual interactions and mechanisms that affect relaxation and identify those processes that contribute to T1 and T2 effects as well as those that affect T2 only. A variety of high resolution spectral techniques at 200-500 MHz, as well as T1 and T2 measurements from 2-500 MHz, will be used. We will measure (a) hydrodynamic effects, or the effects on water intramolecular dipolar interactions, using measurements of deuterium correlation times. (b) cross relaxation between water and macromolecular protons, using proton relaxation in deuterated samples and transient Overhauser effects (c) restricted diffusion in tissues, and its influence on the effective surface area seen by water, using pulse gradient spin echo sequences (d) chemical exchange rates between ionizable protons and water, using the method of Luz and Meiboom (e) diffusion amongst variations in magnetic susceptibility, using various spin echo sequences, theoretical modelling and the method of Karlicek and Lowe (f) the influence of water that is preferentially oriented and rotating anisotropically, using magic angle techniques and the method of Goldburg and Lee (g) paramagnetic interactions by studying the effects of chelates and heteronuclear NOEs. We will study a selected group of proteins, polymers and gels, in different conditions; in aggregates, in free solution or cross-linked and immobilized; in different degrees of denaturation, and in different solvents and buffers wherein the surface character and affinity will be affected. We will thereby assess the effects on water relaxation not only of individual macromolecules and constituents (and thus the significance of alterations in tissue composition alone) but also the role of supra-molecular macroscopic organization, such as molecular association, the formation of organelles, or immobilization in membranes. Measurements in tissues will be correlated with biochemical assays of tissues (protein, lipid, glycogen, water content) and other measures of the characteristics of the constituents. The overall project should provide many new insights into tissue relaxation phenomena to aid in the better understanding of the origin of contrast in NMR images.